We have been able to provide conclusive evidence that the invertebrate stages of Leishmania are fully capable of a sexual cycle during their growth and development in the sand fly vector. The priority of our studies in the past year has been to determine the frequency, timing, and anatomical location of hybrid formation within the midgut, and to seek the existence of a haploid, gamete stage. In addition to drug resistance markers, fluorescent reporter genes have successfully been used for screening hybrid formation. Sorting of cells with dual fluorescence signals has facilitated high throughput screening of a large number of flies and has allowed us to recover hybrids generated at very low frequencies in the sand fly midgut. We have been able to recover hybrids as early as three days post infection, indicating that early developmental stages, e.g. procyclic and/or nectomonad promastigotes, are sexually competent. The fluorescent reporter genes have also been used to successfully screen for backcross hybrids generated between F1 progeny and each parental line. The genetic hybrids are therefore not sterile, and their backcrossing potential will be essential for genetic linkage analyses. The world-wide distribution of Leishmania is determined by the availability of transmission-competent vectors: some support many different Leishmania, while others are highly restricted. This is best exemplified by P. papatasi, which transmit only L. major despite a wide distribution in regions endemic for many Leishmania species. P. papatasi "selectivity" can be reproduced experimentally, and has been attributed to &#946;1,3-linked galactose side chains decorating the abundant L. major surface lipophosphoglycan (LPG) adhesin, which mediate parasite attachment to the P. papatasi midgut to prevent elimination when the digested blood meal is excreted. As geographically diverse L. major display very different LPG galactosylation patterns, we explored the consequences of this pattern diversity to P. papatasi survival. Using natural isolates and L. major lines engineered to express a wide range of LPG galactosylation patterns, we showed L. major survival in P. papatasi PpapJ flies was optimized by expression of highly modified 'mono-galactosylated'LPG and extremely sensitive to LPG side chain length. Surprisingly, L. donovani lines engineered to express a PpapJ-optimal LPG mono-galactosylation pattern did not survive in PpapJ flies, suggesting that additional interactions are required. These studies reveal the fine specificity of Leishmania - sand fly interactions, and the nature of species- and strain-specific parasite molecules that have co-evolved to take advantage of midgut receptors specific to available sand fly vectors. For the purposes of studying pathogenesis and immune response in experimental models, and especially to evaluate experimental vaccines, the use of sand fly transmitted infections rather than needle inocula is preferred, but has only rarely been reproducibly achieved. We have employed experimentally infected, laboratory-reared Phlebotomus duboscqi sand flies to transmit L. major and have attempted to identify environmental conditions and the characteristics of infections that correlate with and predict productive transmission. Transmission of a laboratory adapted strain of L. major, FV1, was poor and could not be improved by manipulating environmental conditions or permitting flies to lay eggs prior to transmission. In contrast, excellent transmissions occurred with a recent field isolate of L. major from Iraq, which in comparison to FV1 generated a significantly higher number and percentage of infectious metacyclic promastigotes in the anterior midgut of infected flies. A numerical cutoff of the number and percent of metacyclics was revealed below which transmission was found unlikely to occur. The findings point to the likely need to maintain the full transmission cycle of laboratory adapted strains in both their vertebrate and invertebrate hosts in order to study the biology of naturally transmitted infections.